The actin cytoskeleton, composed of actin filaments and associated proteins, is an intracellular structure which plays an essential role in such cellular events as changes in cell shape, cell motility, the distribution and stability of integral membrane proteins, and hormone action. We have recently demonstrated that actin filament organization is central to the regulation of epithelial Na+ and cell volume regulatory-K+ channels in epithelial and human melanoma cells, respectively. Our studies have also demonstrated a role for actin phosphorylation as a signaling event in the hormonal regulation of epithelial ion channels. In this application we propose to study the molecular interactions between the actin cytoskeleton and ion channels which are part of a novel signal transduction mechanism. The molecular steps associated with this signal transduction pathway may involve the direct interaction of actin with ion channels, and/or related structures such as actin-binding proteins or plasma membrane lipids. Therefore, we will assess: I) the role of the actin-based cytoskeleton in the control of apical epithelial Na+ channel activity; II) the role of actin phosphorylation as a mediator of actin organization and the regulation of Na+ channel activity; and III) the role of the actin cytoskeleton and, in particular actin-binding proteins such as filamin, in the control of the cystic fibrosis transmembrane conductance regulator (CFTR), which contains putative actin-binding domains. We will utilize either cells in culture expressing Na+ channels and CFTR as well as human melanoma cells with a dysfunctional actin cytoskeleton or melanoma cells genetically rescued by transfection with the cDNA for filamin. We believe these studies will aid in our understanding of a novel signal transduction mechanism for ion channel regulation based on changes in the actin filament organization.